Reactive oxygen species (ROS) are produced in distinct cellular locations - by the organelle location of oxidases and mitochondria - and exert their effects only nanometers from the site of production. Little is known about how cells use and discriminate between plasma membrane generated, mitochondrial, or extracellular sources of reactive oxygen species to control signal transduction. The objective of this application is to investigate ROS spatiotemporal dynamics during T cell signaling through the development of site-specific ROS dyes, high-throughput microfluidic systems, and computational models. We hypothesize that the subcellular sources of ROS create a tightly connected network between mitochondria, endoplasmic reticulum and plasma membrane oxidases to regulate T cell signaling. The rationale for this research is that by understanding when and where ROS is used to target protein oxidation during antigen recognition, cellular oxidation can move from phenomenological observation to a relevant diagnostic biomarker for disease state. In this project we will develop two enabling technologies to facilitate the investigation of site- specific ROS on T cell activation. First, we will create a new membrane-specific dye for detection of superoxide production by NAPDH oxidases. Secondly, we will design microfluidic platforms for single cell manipulation and high-throughput imaging analysis, capable of generating temporally tunable (i.e. oscillatory) stimulations delivering exogenous molecules. These technologies will be used to determine the contributions of localized ROS sources to T cell signaling and investigate spatiotemporal relationships between ROS generation and calcium. Single cell analysis and control systems theory will be used to generate computational models of feedback control between calcium levels and subcellular ROS compartments. The proposed research is innovative because it merges the technological developments of new imaging probes and microfluidic platforms to address the challenge of analyzing local (rather than global) oxidative stress during T cell signaling. The outcomes of this work are expected to fundamentally advance our understanding of how cells use spatially distinct ROS sources to regulate receptor-initiated signaling. This knowledge will have large impact in ultimately redefining intracellular oxidation by more biologically relevant metrics for diagnosis and treatment of diseases.